There are two long-range goals in this renewal application. The first goal is to understand the mechanism by which various classes of cells in mammalian retina obtain energy. The classical view is that glucose is the primary substrate for retinal energy metabolism and that photoreceptor cells have the highest metabolic rates. A different and more recent view (termed "metabolic trafficking") is that Muller cells are the principal, if not sole, producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and retinal neurons. A third preferred substrate, glutamate, initially metabolized by the enzyme aspartate aminotransferase (AAT), has also been proposed. The following specific aims will evaluate these contrasting hypotheses of retinal metabolism. The cellular sites of glucose uptake and phosphorylation will be identified in studies with labeled 2-deoxyglucose. Mitochondrial production of carbon dioxide will be measured with labeled glucose, glutamate, or lactate with and without inhibitors of AAT in order to establish the metabolic priority of substrates utilized by rat and guinea pig retinal mitochondria and the role of AAT in regulating the rate of the Krebs cycle activity. In addition, the specific contribution of Muller cells to glycolysis and respiration in whole retinas will be evaluated by using gliotoxins to selectively kill these cells in rat and guinea pig retinas: cell morphology and activity of glutamine synthetase will be used as markers of toxicity. These experiments will provide new and important information regarding the three competing hypotheses for retinal energy metabolism and provide a basis for a better understanding of the vulnerability of specific retinal ischemia. The second goal expands on these studies of retinal metabolism to understand roles played by ion transport and metabolic trafficking across the RPE in photoreceptor function and retinal adhesion. The proposed experiments were developed following the striking observation that incubation of rat eyecups in bicarbonate-free medium quickly leads to retinal detachment. Experiments will also examine retinal re-attachment by restoration of bicarbonate to the bathing medium or the addition of certain drugs (e.g., diamox) which have been reported to decrease retinal edema. A long-term goal of the second project is to understand better the nature of the interactions between the retina and RPE and factors regulating retinal detachment.